title: Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein.
abstract: Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APE1. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein.
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, Udine, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Tell
authors.forename: Matteo
authors.initials: M
authors.lastname: Di Piazza
authors.forename: Malgorzata M
authors.initials: MM
authors.lastname: Kamocka
authors.forename: Carlo
authors.initials: C
authors.lastname: Vascotto
mesh_terms: D003513:Cycloheximide / Q000008:administration & dosage; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects* / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D018613:Microscopy, Confocal; D009369:Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D010084:Oxidation-Reduction / Q000187:drug effects; D034622:RNA Interference
chemical_list: C583169:Dendra2 protein, Dendronephthya; D008164:Luminescent Proteins; D003513:Cycloheximide; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase